Heretofore it has become common to use metallic particles having superparamagnetic properties to concentrate biological ligands present in small amounts in large volumes of aqueous fluids, including fluids of mammalian origin such as urine. These metallic particles are often of large size (typically in the order of 1–5 μm or larger in average mean diameter) such that they cannot move, or do not move sufficiently readily, through the matrices used for either flow-through tests or lateral flow immunochromatographic (“ICT”) tests such as those commonly used currently in many commercially available diagnostic tests for identifying disease causative pathogens. In an instance where such tests are to follow the initial concentration step, removal of the superparamagnetic particles used for concentration is necessary, followed by adding a target specific conjugate labelled with a chemiluminescent, fluorescent or radioactive tag, or a tag such as colloidal latex particles, colloidal gold, or another colloidal metal which couples to the biological ligand and aids in the detection thereof. The need to remove superparamagnetic particles used in ligand concentration and then subject the concentrated ligand to an identification or quantification assay often poses problems. For example, quantification of the small amount of biological ligand obtained by concentration is rendered inaccurate if even a tiny fragment of concentrated ligand clings to the particles used for concentration; by the same token, incomplete removal of a small fragment of a magnetic particle may disrupt a qualitative identification of the concentrated sample by setting up an interaction with the labelling agent chosen for use in the subsequent identification test. Even in cases where superparamagnetic particles are employed to concentrate biological ligands present in a large volumes of fluid and the nature of the subsequent identification procedure renders separation of the superparamagnetic particles unnecessary, these particles have heretofore-been viewed in the art as irrelevant to the subsequent identification step.
Large sized superparamagnetic particles have been preferred for ligand concentration work, because their large size (in the order of 1 to 5 μm or more) increases the mass of material bound to the target ligand and allows the gradient field of a fixed magnet to effect separation with ease. Much smaller particles have been used in some instances but often the low mass of magnetic material that they impart to their target, requires the introduction of magnetizable columns, filters or screens as an aid to separating the target molecules from the sample.
Particles heretofore used as tags for detecting a biological ligand (regardless of whether it has been subjected to a first concentration step) are usually quite small. As already noted, this is especially true where rapid “flow-through” or lateral flow matrices having narrow pores are employed as solid phase substrates. Particularly in the lateral flow ICT format, particles used as detection markers must be small enough to migrate through the pores of the matrices and reach the immobilized binding partner of the biological ligand being detected.
The present invention is based on the discovery that there is a class of superparamagnetic particles which are small enough to function as tags for detection of biological ligands in ICT test formats where solid porous matrices are employed and also have a sufficiently large magnetic moment to function effectively as ligand concentration adjuvants. The capability of using the same particles for concentration and separation of a target ligand from a large volume of liquid and as tags for a qualitative ligand identification test or a similar test that not only identifies but quantifies the amount of ligand enables a significant increase in the sensitivity of the pre-assay concentration step. At the same time, the separation of the target ligand from interfering or inhibitory substances that may be present in the original sample is enhanced, the awkward need for removing a magnetic label is avoided and so is the equally awkward need for introducing a second label.